High efficiency protein extraction

ABSTRACT

The invention relates to a process for isolating edible protein from animal muscle by solubilizing the protein in an alkaline aqueous solution.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US01/27513, filed Sep. 5, 2001, which claims the benefit of priorityfrom U.S. Provisional Application No. 60/230,397, filed Sep. 6, 2000.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under U.S. Department ofCommerce, National Oceanic and Atmospheric Administration Grant No.5700000741; and U.S. Department of Agriculture, National ResearchInitiative Competitive Grant Program, Grant Nos. 9701691 and99-33503-8285. The Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to a process for isolating edible protein fromanimal muscle by solubilizing the protein in an alkaline aqueoussolution.

BACKGROUND OF THE INVENTION

Surimi or formed fish has been produced in Japan for about a thousandyears. Only recently has surimi appeared in North American supermarketsas imitation crab legs, lobster chunks, shrimp, and scallops. NorthAmerican surimi is typically produced from lean white fish, such aspollock or whiting.

Low value animal muscle (e.g., from fatty pelagic fish or poultry boneresidue) is usually undesirable as a source of food for humanconsumption. After processing, the isolated protein is oftencharacterized by unattractive textures, dark colors, and strong flavors,often as a consequence of membrane lipid oxidation.

SUMMARY OF THE INVENTION

The invention is based on the discovery that if animal muscle protein issolubilized in an alkaline solution, the resulting soluble protein canbe isolated in high yields and in a substantially native andnon-oxidized form more suitable for human consumption. It was discoveredthat alkaline treatment of animal muscle minimized the oxidative effectsof deoxyhemoglobin and the hydrolysis of myosin, a major muscle protein,by lysosomal proteases. After the muscle protein is solubilized in analkaline aqueous solution, various undesirable components (e.g., bones,neutral lipids, membrane lipids, fatty pieces, skin, cartilage, andother insoluble material) can be removed. The soluble protein is thenprecipitated and collected in an edible form.

Accordingly, the invention features a method for isolating edibleprotein from animal muscle (e.g., fish, such as pelagic fish, orchicken) by obtaining a mixture comprising animal muscle and water;increasing the pH of the mixture to a level sufficient to solubilize atleast a portion of the insoluble animal protein in the animal muscleprotein mixture; removing at least about 50% by weight of total membranelipids from the mixture; precipitating the solubilized protein from theanimal muscle protein mixture; and collecting the precipitated protein,thereby isolating the edible protein from the animal muscle. Thisisolated protein can be used for forming edible protein gels that can beused in foods such as, e.g., hot dogs and cooked surimi. To furtherlimit the extent of oxidation, especially of membrane lipids, themixture can include an iron chelator (i.e., a compound that binds to andinactivates the oxidizing potential of an iron atom or ion), such asethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacidicacid (DTPA), carnosine, anserine, uric acid, citric acid, phosphate,polyphosphate, ferritin, or transferrin

The method can include an optional washing step, in which the raw animalmuscle is rinsed with water prior to solubilization; or a step forremoving insoluble matter such as bone skin and cartilage fromsolubilized protein. This can be accomplished using an optionallow-speed centrifugation step prior to precipitation of the protein. Asused herein, “low-speed” means about 4000×g or lower (e.g., 2000, 2500,3000, 3250, 3500, or 3750×g), and “high-speed” means about 5000×g orhigher (e.g., 5500, 6000, 7500, 8500, 10,000, or higher×g).Centrifugations can be performed for a sufficient time (e.g., 5, 10, 15,20, 25, 30, 40, 60, or more minutes) to achieve the intended result,such as removal of membrane lipids or removal of insoluble material fromthe mixture.

The animal muscle can in general constitute 50% or less (e.g., 40, 30,20, 15, 10, or 5% or less) by weight of the mixture. When removal ofmembrane lipids from the soluble proteins is desired, the percentage ofanimal muscle in the mixture should be less, e.g., 15, 10, or 8% or lessby weight of the mixture, to render the viscosity of the solution lowenough for separation of membrane lipids from an aqueous portion of themixture. When the viscosity of the solubilized protein is reduced, atleast about 50% (e.g., at least about 60, 70, 80 or 90%), by weight ofthe total membrane lipids present in the mixture can be removed.

Membrane lipids can be removed from a mixture using a number of methods.For example, centrifugation of the mixture at about 5000×g or higher(e.g., 6000, 7000, 8000, 9000, or 10,000×g or higher) is sufficient topellet the membrane lipids below an aqueous layer containing solubilizedprotein. Where necessary or desirable, neutral lipids (e.g., oils) canbe removed from the top of the aqueous layer. Other methods of removingmembrane lipids from the mixture include filtration and the addition ofan aggregant. As used herein, an “aggregant” is a material that, whenadded to a mixture, causes one or more dispersed components of themixture to aggregate, thereby facilitating separation of the one or morecomponents from the mixture.

The initial solubilization of animal muscle protein can be accomplishedby increasing the pH of mixture to about 10.0 or above (e.g., 10.5 orabove). The pH can be increased by adding polyphosphate to the mixture.

The solubilized protein can be precipitated by lowering the pH of thealkaline mixture to, e.g., about 5.5 or lower. For example, the pH canbe lowered to about 4.0 or below (e.g., 2.5 to 3.5, especially 3.0),then raised to about 5.0 or above. The pH of the aqueous phase can bedecreased by adding an acid, e.g., hydrochloric acid, to the aqueousphase. The salt concentration can be optionally adjusted to aidprecipitation (e.g., by adding a salt such as NaCl), and acryoprotectant optionally added to the precipitated protein. Theprecipitated protein can be collected by centrifugation and/or with theaid of an aggregant, such as a polyamine (e.g., spermine or spermidine),a neutral or ionic polymer, or any other specific aggregant that is alsouseful for aggregating membrane lipids.

In another aspect, the invention includes a method of isolating edibleprotein from animal muscle (e.g., fish or chicken) by obtaining amixture comprising animal muscle and water; increasing the pH of themixture to a level sufficient to solubilize at least a portion of theinsoluble animal protein in the animal muscle protein mixture;precipitating the solubilized protein from the animal muscle proteinmixture; and collecting the precipitated protein, thereby isolating theedible protein from the animal muscle. In this method, the temperatureof the mixture is maintained at 15° C. or less (e.g., 10° or 5° or less)in each step to minimize denaturing of the protein and deleteriousoxidation of contaminants such as membrane lipids. The collectedprecipitated protein provides a yield of at least 70% (e.g., at least80, 90, 95%) by weight of the total animal muscle protein in the mixtureprior to increasing the pH. Additional optional steps and materials, asdescribed herein, can be used in this method, where applicable.

As an alternative to increasing the pH of a mixture containing animalmuscle and water, the animal muscle can first be obtained and then mixedwith an aqueous solution having a pH sufficiently alkaline to solubilizeat least a portion of the animal protein.

The invention has several advantages. The methods of the inventioninactivate or reduce the oxidative potential of hemoglobin, as well asminimize hydrolysis of myosin, a major component of animal muscle. Inaddition, optional features of the invention remove essentially all ofthe membrane lipids, thereby further stabilizing the edible proteinagainst oxidation. Thus, the invention embodies a strategy ofinactivating oxidants and removing undesirable substrates for oxidation,both of which help render an edible protein product suitable forcommercial food products.

The methods described herein are useful for processing fatty muscletissues as a feed composition, which are typical of low cost rawmaterials, such as would be found in the fatty fish species ormechanically deboned poultry meat. In addition, the methods are usefulfor isolating edible protein from lean animal muscle, such as white fishmeat (e.g., cod).

The process of this invention also provides for increased yield ofprotein from animal muscle. Greater than about 70% by weight of proteincan be typically obtained from muscle tissue using the methods of theinvention. In some cases, protein yields of greater than 90% by weightcan be achieved. Besides the obvious commercial value of having betteryields, the improved yield results in less protein in the waste waterduring industrial processing, so that environmental pollution isdecreased.

The methods of this invention do not require fresh or lean animal muscleas a starting material. Any spoilage (off smells or colors) due tooxidized lipids can be removed using the new methods. In addition,animal parts containing other fatty tissues such as skin can be used,since the offending lipids, as well as the parts themselves, can beremoved. In the case of fish processing, the new methods eliminate theneed to fillet the fish prior to protein isolation, thereby reducing thecost of processing. Similarly, by removing the lipids, the methods ofthe present invention reduce the amount of fat-soluble toxins (e.g.,polychlorinated biphenyls or PCBs) in the food product.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable methods andmaterials for the practice or testing of the present invention aredescribed below, other methods and materials similar or equivalent tothose described herein, which are well known in the art, can also beused. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a bar graph of the amount of thiobarbituric acid reactivesubstances (TBARS) in cod muscle at specific pH values. The “pH 3 to 7”bar was held at pH 3 for 1 hour after the addition of hemolysate, beforeadjusting to pH 7 and then storage.

DETAILED DESCRIPTION

The invention relates to a new method of isolating edible protein fromanimal muscle. The resulting edible protein is relatively free fromoxidation products, is capable of forming a gel, and can be processedinto human foods. For example, the methods of the invention can be usedto produce surimi from fatty fish as well as leaner white fish.

I. Isolating Lipid-Free Edible Protein

In general, the invention features a method for isolating edible proteinfrom animal muscle (e.g., fish or chicken muscle) by first obtaining amixture containing animal muscle and water, the animal muscle can beless than about 15% (e.g., 5% to 12%, or 10%) by weight of the mixture.Any aqueous solvent, e.g., water, can be used. In addition, the musclecan be washed with an aqueous solution prior to any mechanicalmanipulation. The muscle can be substantially diluted in water such thatthe solubilized protein suspension/solution produced in successive stepsof the method is of a low enough viscosity so that the lipids orinsoluble material can be removed by centrifugation. Lower viscosity canalso aide removal of mixture components using methods other thancentrifugation, as described herein. The viscosity of the proteinsuspension/solution is preferably about 75 mPa·s or less (e.g., about 35mPa·s or less). Viscosity is measured, for example, with a BrookfieldModel LVF viscometer (Brookfield Engineering, Stoughton, Mass.) using a#3 or #4 spindle at 60 rpm. The manufacturer's supplied conversion chartis then used to calculate viscosity. The animal muscle can bemechanically ground, homogenized, or chopped by hand.

After dilution of the animal muscle with water or an aqueous solution,the pH of the mixture is then increased, for example, to greater thanabout 10.0 (e.g., about 10.0 to 11.5, or about 10.5) so that at least50%, e.g., at least 60, 70, 75, 80, 85, or 90%, of the animal protein byweight is solubilized. Alternatively, an aqueous solution containingsufficient base to raise the pH of the mixture to greater than about10.0 (e.g., about 10.0 to 11.5, or about 10.5) can be added to theanimal muscle to achieve the same level of solubilization.

Protein denaturation and protein hydrolysis is a function of temperatureand time in solution, with increasing temperature and time in solutionpromoting protein denaturation and hydrolysis. Thus, it is desirable toreduce the temperature and the time the protein is in solution. As aresult, the methods of the invention are preferably conducted at about0° C. to 10° C. (e.g., 0° C., 1° C., 4° C., or 6° C.). The methods ofthe invention can also be carried out using frozen starting material,e.g., frozen muscle tissue. The aqueous composition also may containcomponents such as preservatives, which protect proteins fromdegradation. The ionic strength of the solution can be adjusted to avoidprotein precipitation. Muscle tissue can also be homogenized, e.g.,broken into pieces of approximately 5 mm or less, to achieve rapidextraction on adjustment of the pH, to further prevent denaturation ofthe proteins.

To remove membrane lipids from solubilized protein, the mixture can becentrifuged (e.g., at from about 5000×g to 10,000×g, or higher), so thatthe charged membrane lipids are separated from an aqueous phase, whichis collected by, for example, decanting the aqueous phase. Severallayers can form after centrifugation. At the bottom, the chargedmembrane lipids and any remaining residue is pelleted. The percentagesediment weight can be less than 20% (e.g., less than 10%), because ahigher sediment percentage indicates that some of the desirable proteinhas been removed with the undesirable lipids. Percentage sediment weightis defined as the weight of pellet after centrifugation divided by thetotal homogenate weight. Above the pellet is an aqueous layer containingthe solubilized protein. At the top, the neutral lipids (fats and oils),if any, float above the aqueous layer. The neutral lipids can be removedwith a pipette before decanting the aqueous phase. Intervening layerscan also be present depending on the source of muscle. For example, agel of entrapped water containing solubilized protein can form betweenthe aqueous layer and the pellet. This gel can be kept with the aqueouslayer to increase protein yield. Of course, in industrial applications,the aqueous phase (and other phases, if desired) can be removed duringcentrifugation using a continuous-flow centrifuge or other industrialscale machinery.

Other methods besides centrifugation can be used to separate themembrane lipids from the aqueous phase. For example, a variety offiltration apparatus are available to the skilled artisan, depending onthe size and volume of the material to be separated. In the absence ofmembrane lipid aggregants, a microfiltration apparatus is suitable forseparating molecules having molecular weights in the range of 500,000 to20 million. If the lipids are aggregated, particulate filtration may besuitable. These filtration units typically operate under pressure in therange of 2 to 350 kPa. In addition, cationic exchange membranes (sc-1)and anionic exchange membranes (sa-1) are suitable for removing membranelipids from the mixture. In addition, various filtration methods can beused to select for or against muscle proteins of a particular size.

In some circumstances, an HF-lab-5 ultrafiltration unit (Romicon, Inc.,Woburn, Mass.) can be used with a feed tank having an immersed coolingcoil to maintain a relatively constant temperature. A cross flowprocess, which has the advantage of removing filter cake continuously,can also be used. To recover water or lower the salt content of themixture, filtration membranes can be used with electrodialysis to driveout ions from the mixture. For this particular purpose, a stackpack unit(Stantech, Inc., Hamburg, Germany) can be used. This unit containsseveral cell pairs sandwiched between two electrode compartments.

Removal of membranes can also be facilitated by subjecting a mixture tohigh pressure, using, e.g., the MPF 7000 device (Mitsubishi HeavyIndustries, Ltd.) or the High Pressure ACB 665 device (Gec, Alsthom;Nantes, Frances). High pressure treatment, accompanied by the propertemperature treatment, has the added benefit of killing known pathogens,in addition to membrane lipid aggregation and separation.

In addition to the use of high pressure, an aggregant can also be addedto the mixture to facilitate membrane lipid removal. Suitable polymeraggregants include carrageenan, algin, demethylated pectin, gum arabic,chitosan, polyethyleneimine, spermine, and spermidine. Other aggregantsinclude salts, such as a calcium salt, magnesium salt, sulfate,phosphate, and polyamine.

The pH of the aqueous phase can then be decreased so that thesolubilized proteins precipitate. The yield can be at least 70% (e.g.,at least 90%) by weight of the total starting protein in the mixture.The yield is defined as the precipitated protein mass divided by thetotal muscle protein mass. In one embodiment, the pH is decreased toabout 5.5 or less to precipitate and collect the protein by, forexample, centrifugation. In another embodiment, the pH of the aqueousphase is decreased to less than about 4.0 (e.g., about 2.5 to 3.5, orabout 3.0) and then increased to more than about 5.0 to precipitate theprotein. This further dip in pH may facilitate precipitation ofsarcoplasmic proteins at the higher pH. Cryoprotectants (e.g.,disaccharides and/or polyalcohols, such as polysorbatol) can be added tothe precipitated protein to preserve and protect the product duringfreezing and storage.

Any acid that does not undesirably contaminate the final product can beused to lower the pH of the centrifuged mixture. For example, organicacids (e.g., malic acid, or tartaric acid) or mineral acids (e.g.,hydrochloric acid or sulfuric acid) are suitable. Citric acid which hasa favorable pK_(a) value can provide buffering capacity at pH 3 and pH5.5. Acids that have significant volatility and impart undesirableodors, such as acetic acid or butyric acid, are undesirable. Likewise,any of several bases can be used to raise the pH. A polyphosphate issuitable, since it also functions as an antioxidant and improves thefunctional properties of the muscle proteins.

Since the control of the pH of a mixture can often be difficult, themixture can include a buffer that maintains an acidic target pH value ora basic target pH value. For example, a compound such as citrate, whichhas a pK_(a) in the range of about 5.97, can be added to the mixturecontaining solubilized protein, if the solubilized protein is to beprecipitated at a pH of about 6.0 or lower. In effect, citrate can actas a “brake” to ensure that the pH of the mixture does not over-shoot atarget pH value. Given a target pH, the choice of buffer is within theskill in the art of food science. Buffers suitable for a target pH inthe range of 8.0 to 9.0 include glycine, arginine, asparagine, cysteine,carnosine, taurine, pyrophosphate, and orthophosphate. Buffers suitablefor a target pH in the range of 5.5 to 6.5 include histidine, succinate,citrate, pyrophosphate, and malonate. Buffers suitable for a target pHin the range of 2.0 to 2.5 include alanine, glutamic acid, citric acid,lactic acid, phosphoric acid, or pyruvic acid.

Instead of reducing the pH of the solution, protein precipitation can beattained by adding polymers such as polysaccharides, charged polymers,marine hydrocolloids including alginates or carrageenan or the like,either alone or in combination with centrifugation. The saltconcentration of the aqueous phase can also be adjusted to facilitateprecipitation.

In addition, the various washes, supernatants, and flow-throughfractions can be recycled back to earlier steps to recover even moreprotein using the methods. For example, after the solubilized proteinhas been precipitated, the aqueous fraction can be entered into anotherbatch of animal muscle that has yet to be solubilized.

II. Use of Lipid-Free Edible Protein

The new methods can be used to process for human consumption materialsthat are not presently used as human foods because of their instabilityand unfavorable sensory qualities. Small species of fish such asherring, mackerel, menhaden, capelin, anchovies, or sardines are eitherunderutilized or used for nonhuman uses. Approximately one half the fishpresently caught in the world are not used for human food. The newmethods allow for better utilization of the available food supply. Themethods can utilize both white-flesh and dark-flesh fish, as well aschicken and other materials. The quality of non-fatty animal muscle(e.g., cod) can be improved in terms of yield using the methods of theinvention. The methods of the present invention result in proteinisolates that are capable of forming gels, e.g., gels from mechanicallydeboned chicken meat, that are stronger than gels made from materialsnot processed by the methods of the present invention. Further, the gelshave reduced fat and increased water binding ability compared to gelsmade from unprocessed materials. Further, the protein isolates producedby the methods of the present invention can be used as a functionalingredient to replace protein portions, e.g., meat, of various foodproducts, such as sausages.

III. Sources of Animal Muscle

The process of this invention can be used to process flesh that isrecovered from fish after the fillets have been removed. This materialis typically not used for human food. Similarly, there is very littleusage of the skeletons of chickens after parts are removed for retailsale. The methods of the present invention can process such chicken andfish parts to produce edible protein suitable for human consumption.Other underutilized muscle sources useful in the methods of theinvention include Antarctic krill, which is available in largequantities but is difficult to convert to human food because of itssmall size.

Representative suitable starting sources of animal muscle for theprocesses of this invention include fish fillets, deheaded and guttedfish, crustacea (e.g., krill), molluscs (e.g., squid), chicken and otherpoultry (e.g., turkey), beef, pork, or lamb.

The invention will be further described in the following examples, whichdo not limit the scope of the invention defined by the claims.

EXAMPLES Example 1 Titrating pH for Optimal Protein Solubilization

Preparation of fish. Excellent quality Atlantic cod was obtained fromlocal fish processors. Cod muscle was well trimmed, ground to ⅛-inchpieces, mixed with nine parts cold (6° C.) deionized, distilled waterfor each part muscle, and homogenized in a Polytron® PCU 1 machine(Brinkman Instruments, Westbury, N.Y.) at a speed of 76 for 1 minute.

Alkaline solubilization. The pH of the cod homogenate was 6.85. Onemolar NaOH was added to the homogenates until it reached specificalkaline pH levels in the range of 9.04 to 11.50. The viscosities of thesolutions at 4-6° C. at the specific pH values were measured with aBrookfield Model LVF® viscometer (Brookfield Engineering, Stoughton,Mass.) using a #3 or #4 spindle at 60 rpm. The manufacturer's suppliedconversion chart was used to calculate viscosity. The mixture was thencentrifuged at 9300 rpm in a No. 35 rotor (10,000×g) for 60 minutesusing an L5-65B® ultracentrifuge, to form a top layer of emulsified oil,a middle aqueous layer containing the solubilized protein, and amembrane pellet. In some cases, when lean white fish is used, theemulsified oil layer may not be present. The aqueous layer was collectedby removing the oil with a pipette and then decanting the aqueoussolution. The viscosity and solubility results are shown in Table 1.

TABLE 1 viscosity % protein % sediment pH (mPa · s) solubility weight9.04 373.5 33.37 31.18 9.50 409.0 36.85 40.42 10.00 638.5 78.82 28.2210.49 59.5 88.90 15.08 10.99 57.4 99.56 13.52 11.50 29.5 >99.9 4.95 6.85222.5 — —Protein mass was determined by using the Biuret reaction as described inTorten et al., J. Food Sci. 168:168-174, 1963. The percentage proteinsolubility is defined as protein mass in the aqueous layer divided byprotein mass in the original homogenate. The percentage sediment weightis the weight of sediment after centrifugation divided by the totalhomogenate weight. High sediment weight values are indicative of proteinremoved with the membrane lipids. The bottom row in Table 1 representsthe homogenate prior to adjustment with 1 M NaOH.

Table 1 indicates that greater than 70% protein solubility occurs at pHvalues above 10.0, viscosity drops below 75 mPa·s at pH values between10.0 and 10.5 and above, and percentage sediment weight drops below 15%at about pH 10.5 or above. The data in Table 1 show that efficientprotein solubility (>70%) occurs at pH values above about 10.5. As theviscosity drops below 75 mPa·s when the pH is above about 10.5, thepercent protein solubility increases to above 75%. Similarly, percentagesediment weight decreases to below 15% when the pH rises above 10.5. Ifthe viscosity was too high, the protein co-sedimented with the membraneand was removed. A viscosity of 75 mPa·s or less was typically needed toremove the membrane lipids by centrifugation, without removing asubstantial portion of the protein along with them. The sample at pH 10was highly viscous with good protein solubility. This sample, however,would have been difficult to work with in an industrial setting.Sediment weight percentages of about 15% or lower was consideredacceptable. Thus, although a pH of about 10.0 could be used, higher pHvalues approaching and above 10.5 were of greater commercial interest.

Example 2 Production of Cod and Mackerel Surimi

Cod was prepared as described in Example 1 above. Atlantic mackerel wasalso obtained from local fish processors and processed as described inExample 1. The mackerel was of Stage II quality as assessed using themethod described in Kelleher et al., J. Food. Sci. 57:1103-1108 and1119, 1992. The mixtures were adjusted to pH 10.5 to solubilize theprotein. The mixtures were then centrifuged, and the aqueous layercollected as described in Example 1.

One molar HCl was added to the aqueous protein solution until it reachedpH 5.5. The precipitated protein was collected by centrifuging at 15,000rpm (34,600×g) in a No. 19 rotor for 20 minutes in a Beckman® L5-65Bultracentrifuge. The supernatant was decanted. A cryoprotectant solutioncontaining 4% sucrose, 4% sorbitol, and 1.2% sodium tripolyphosphate wasadded to the protein pellet. The mixture was formed into surimi bychopping for 30 seconds using an Oskar® model chopper (Sunbeam-Oster,Hattiesburg, Miss.) in a refrigerated, walk-in cooler. The surimi waspacked into polyethylene Whirl-pak7® bags and frozen at −40° C. for atleast 12 hours.

The frozen surimi was tempered in a walk-in cooler (4° C.) for 30minutes prior to chopping for 2 minutes in the Oskar® chopper. NaCl wasadded to 3% (w/w) of surimi during chopping. The chopped paste wasstuffed into stainless steel tubes (19 mm diameter×175 mm) and cooked at90° C. for 20 minutes. The cooked surimi was set in ice for 20 minutesprior to being discharged from the tubes and held for 24 hours at 6° C.Physical properties of the cooked food product are shown in Table 2. Gelstrength and displacement values were determined using a 5 mm stainlesssteel probe attached to an Instron® Model 1000 Universal MaterialsTesting Instrument (Instron Corp., Canton, Mass.) equipped with a 5 kgload cell and a crosshead speed of 100 mm/min. The values were recordedand calculated as described in Lanier, “Measurement of SurimiComposition and Functional Properties,” In: Surimi Technology (Lanier etal., eds.), pp 123-163, Marcel Dekker, Inc., New York, 1992.

TABLE 2 Muscle Source Strain Stress (kPa) Cod 2.21 ± 0.10 128.13 ± 7.33Mackerel 1.95 ± 0.08  91.2 ± 0.00For cod, the values represent the average and standard deviation ofthree cooked tubes from one gel sample. For mackerel, the valuesrepresent the average and standard deviation of two cooked tubes fromone gel sample.

All gels were of good quality. In general, values of strain (elasticcomponent) greater than 1.9 to 2.0 are rated as grade A gels. Stress(hardness component) values found in all gels were excellent, with mostcommercially available gels being at least about 30-35 kPa.

Example 3 Production of Protein Isolates from Herring Light Muscle

Preparation of fish. Fresh herring was obtained from D&B Bait,Gloucester, Mass., and transported on ice to the University of Mass.Marine Station (approx. 15 min. travel time). Upon arrival in thelaboratory, the fish was graded visually and divided into four grades:In rigor, stage I, II, and III (Kelleher et al., J. Food. Sci.57:1103-1108 and 1119, 1992). The post mortem age generally rangedbetween 6-36 hours. White muscle was manually excised and pushed througha 3 mm plate using a kitchen grinder (Kitchen Aid Inc., St. Joseph,Mich., USA).

Protein isolation. Ground muscle (120-300 g) was homogenized for 1minute (speed 50, 120 V) with 9 volumes of ice-cold distilled waterusing a Kinematica Gmb H Polytron (Westbury, N.Y., USA) connected to aVariable autotransformer (Dayton, Ohio USA). The proteins in thehomogenate were solubilized by drop-wise addition of 2N NaOH untilreaching pH 10.8. The protein suspension was centrifuged within 15minutes at 18,000×g (20 minutes) giving rise to four phases: a floating“emulsion layer,” a clear supernatant, a soft gel-like sediment, and aslightly harder bottom sediment. The supernatant was separated from the“emulsion layer” by filtering these two phases through doublecheesecloth. The soluble proteins were precipitated by adjusting the pHto values between pH 4.8 and 7, e.g., 5.5, using 2N HCl. Precipitatedproteins were collected via a second centrifugation at 10,000×g.Manufacture of surimi. Excess water in the alkali-produced proteinprecipitates was squeezed out manually or removed via centrifugation (20minutes, 18,000×g). This lowered the moisture content (Mc) of thealkaline produced precipitates from 88±1% to 72±3% (n=7). Bothprecipitates were then adjusted to 80% Mc with distilled water andblended with the cryoprotectant mixture (4% sucrose, 4% sorbitol, 0.3%sodium tripolyphosphate). The final Mc was 73.2±0.5%. The surimi wasfrozen in plastic bags at 80° C.

Manufacture of surimi gels. Gels were prepared as described by Kelleher& Hultin, (Kelleher & Hultin, Functional Chicken muscle protein isolatesprepared using low ionic strength and acid solubilization/precipitation,In Meat Science in the New Millennium, Procedings from the 53^(rd)annual reciprocal meat conference, The Ohio State Univeristy, Jun.18-21, pp 76-81 (2000)). with the exception that the pH of the surimiwas adjusted to 7.1-7.2 using 10% NaOH or 10% HCl after chopping in 2%NaCl. Surimi was packed either in cellulose casein (The Sausage MakerInc., Buffalo, N.Y.) or in 19 mm metal tubes, depending upon the type ofgel measurements to be carried out.

Quality of gels. Strain and stress (at structural failure) were analyzedusing the torsion technique of Wu et al., J. Tex. Studies, 16: 53-74(1985), or with a Rheo Tex model gelometer AP-83 (Sun Sciences Co.Seattle, Wash., USA). The latter measured the deformation (mm) and thepeak force (g) required to penetrate 2.5 cm sections of the gels. Gelswere also subjected to the folding test described by Kudo et al. (1973)by folding a 3 mm slice of the gel once or twice. The Hunter colorvalues, “L,” “a,” and “b,” were measured on gels according to Kelleherand Hultin. Supra.

Table 3 provides data from alkali aided preparation of surimi and surimigels using fresh herring light muscle and herring light muscle from fishaged 6 days on ice, which was processed in the same way. Thecryoprotectant mixture consisted of 4% sorbitol, 4% sucrose, and 0.3%sodium tripolyphosphate. Gels contained 2% NaCl and were formed at 90°C. for 30 minutes. Break force and deformation were measured with aRheotex AP-83 (Sun Science Co. Ltd, Nichimo International Inc, Seattle,Wash., USA). Values within the same row bearing different numbers aresignificantly different (p≦0.05). The data indicate that good qualitysurimi and surimi gels can be prepared from both fresh and aged herringusing the methods of the present invention.

TABLE 3 Fresh herring Aged herring pH 10.8 pH 10.8 Raw material/surimicharacteristics Moisture Content (Mc) in muscle (%) 79.6 80.6 Musclelipid content (% dw) 11.1 8.8 Muscle TBARS (μmol TBA/kg) 5 28 Mc inprotein precipitate (%) 87.3 87.7 Mc in dewatered protein precipitate(%) 74.4 74.5 Mc in surimi with cryoprotectants 72.5 73.1 pH in surimiwith cryoprotectants 6.87 6.42 pH prior to gelation 7.15 7.11 Mc infinal surimi gel (%) 70.7 69.9 Gel characteristics Folding test 5 3Break force (g) 871 ± 62 464 ± 11 Deformation (mm)  9.2 ± 0.7  6.2 ± 0.3

Table 4 provides data from alkali aided preparation of surimi and surimigels from fresh herring light muscle. The cryoprotectant mixtureconsisted of 4% sorbitol, 4% sucrose, and 0.3% sodium tripolyphosphate.Gels contained 2% NaCl and were formed at 90° C. for 30 minutes. Stressand strain was measured using the torsion technique (Wu et al., J. Tex.Studies 16: 53-64 (1985)) using a Brookfield Digital viscometer (ModelDV-II, Brookfield engineering Inc. Stoughton, Mass., USA). Results areexpressed as mean±SD (n=4). Color was measured with a Hunter LabScan IIcolorimeter (Hunter Associates Laboratories, Reston, Va.). Colormeasurements (are expressed as mean±SD (n=5). Whiteness was calculatedaccording to the following formula: 100−((100−L)²+a²+b²)^(0.5) (Lanier,“Measurement of Surimi Composition and Functional Properties,” In:Surimi Technology (Lanier et al., eds.), pp 123-163, Marcel Dekker,Inc., New York, 1992) using the average values of L, a, and b (SeeKelleher and Hultin (2000), Supra).

TABLE 4 pH 10.8 Raw material/surimi characterisitics Mc in muscle (%) 80Muscle lipid content (% dw) 11.3 Mc in protein precipitate (%) 87.5 Mcin dewatered protein precipitate (%) 72.8 Mc surimi with cryoprotectants(%) 73.6 pH surimi with cryoprotectants 6.0 pH prior to gelatin 7.1 Gelcharacteristics Mc in final surimi gel (%) 74.1 Folding test 5 Stress(kpa) 56.1 ± 2.4 Strain  1.6 ± 0.1 G 35.4 ± 2.1 L 66.5 ± 0.3 a −2.4 ±0.4 b  8.1 ± 0.9 Whiteness 65.5

Example 4 Production of Protein Isolates from Mechanically SeparatedDeboned Chicken Meat (MSDC)

A protein isolate was prepared from MSDC by the alkaline process similarto that described in Example 3. The protein isolate was collected at pH5.5. The protein isolates were then divided into two batches and 2.5%NaCl was added to each batch. The pH of one sample was adjusted to 6.0,and the other was adjusted to pH 7.0. The material was then stuffed intocasings and heated in a water bath for 30 minutes at 90° C. The materialwas then removed, cooled in an ice bath, and stored overnight in arefrigerator before testing. Gels were also prepared directly from MSDCas a control.

The gelation characteristics of the protein isolate (prepared by themethod of the present invention) and the original MSDC were compared.Results are provided in Table 5.

TABLE 5 Protein Isolate Protein Isolate pH 6.0 pH 7.0 MSDC % lipidoriginal (dry basis) — — 52.2 % lipid (dry basis 9.1 9.5 41.7 pH 6.187.03 6.66 % water 78 79 64 L value 54 52 48 Torsion test Stress (kPa) 78— 44 Strain 1.45 — 1.45 Puncture, gel strength, g.cm Less heating^(a)677 463 395 More heating^(a) 842 517 255 ^(a)Exposed to cookingtemperature for a shorter or longer period because of location insample.

Protein isolates prepared by the method of the present invention showedimprovements in water binding and in gel strength. The lean portion ofthe protein isolate prepared at pH 6 had 28% more water (and 21.1%greater weight) than the lean portion of the MSDC. The protein isolatesalso had a lower lipid content than the MSDC.

Example 5 Use of a Protein Isolate as a Functional Ingredient, and theEffect of Various Chopping Methods on Gel Quality

The effect of utilizing a protein isolate prepared by the methods of thepresent invention as an ingredient in foods was investigated.Specifically, alkaline-extracted protein isolates were substituted forchicken breast muscle in chicken breast muscle wieners. Further, theeffect of various chopping methods on gel quality was investigated.

Preparation of Protein Isolate. 4800 ml of water was added to 600 g ofMSDC (1:8 w/v). The mixture was homogenized with a Polytron for 2minutes and the fat at the top of the mixture was removed. The pH wasthen adjusted to 10.5. The mixture was centrifuged at 10,000×g for 30minutes. The neutral fat at the top of the mixture and the insolublefraction in the sediment (which contains mostly collagen and boneresidue) was removed. The supernatant was passed through a double layercheesecloth to retain the fat globules, and the pH was adjusted to 5.5to precipitate the protein. The mixture was then centrifuged twice at10,000×g for 30 minutes. The sediment was centrifuged again at the samespeed for 30 minutes to further reduce the moisture content.

Preparation of Wieners. Wieners containing 0%, 25%, and 50% proteinisolate (PI) were prepared according to the formulae shown in Table 6.

TABLE 6 Control 100% 25% PI 50% PI Ingredients CBM SubstitutionSubstitution CBM 124.13 93.09 62.06 PI 0.00 31.03 62.06 Ice 12.41 12.4112.41 Salt 2.89 2.89 2.89 STP 0.49 0.49 0.49 Na Nitrite 0.02 0.02 0.02Erythobate 0.07 0.07 0.07 Fat (pork, 30%) 60.00 60.00 60.00

Chopping Methods. Chopping methods utilized were as follows: method (a):a mixture containing all ingredients was chopped for 2.5 minutes; method(b): a mixture containing all ingredients except fat was chopped for 1minute, followed by the addition of fat and additional chopping (1.5minutes); method (c): a mixture containing all ingredients except fatand protein isolate was chopped for 1 minute, followed by the additionof fat and additional chopping (0.5 minutes), followed by the additionof protein isolate and additional chopping (1 minute). Mixing by handwas performed for every 30 seconds of chopping, at a temperature of lessthan 18° C. Results are depicted in the tables below.

TABLE 7 Percent water loss after cooking and cooling % Total WaterProtein Composition Chopping Method % Total Fat Loss Loss 100% CBM a NotObserved 3.8 ± 0.6 75% CBM + 25% PI a Not Observed 4.5 ± 0.7 75% CBM +25% PI b Not Observed 4.3 ± 0.4 75% CBM + 25% PI c Not Observed 4.5 ±0.3 50% CBM + 50% PI a Not Observed 7.5 ± 0.6

TABLE 8 pH values of gel product Protein Composition pH 100% CBM 6.2775% CBM + 25% PI 6.21 75% CBM + 25% PI 6.19 75% CBM + 25% PI 6.20 50%CBM + 50% PI 6.18

TABLE 9 Torsion test Protein Composition Stress Strain 100% CBM 81.1 ±4.9 1.68 ± 0.02 75% CBM + 25% PI 80.8 ± 3.8 1.66 ± 0.06 75% CBM + 25% PI 97.5 ± 10.7 1.78 ± 1.72 75% CBM + 25% PI 88.9 ± 8.9 1.72 ± 0.11 50%CBM + 50% PI 76.22 ± 9.9  1.46 ± 0.11

TABLE 10 Color comparison Protein Composition L a b 100% CBM 78.44 ±0.45  4.7 ± 0.68 10.43 ± 0.64 75% CBM + 25% PI 73.22 ± 0.48 7.47 ± 0.1910.48 ± 0.05 75% CBM + 25% PI 73.72 ± 0.35 7.43 ± 0.17 10.59 ± 0.17 75%CBM + 25% PI 71.64 ± 0.88 8.28 ± 0.18 10.61 ± 0.18 50% CBM + 50% PI70.03 ± 0.54 7.29 ± 0.14 10.16 ± 0.10

It was observed that at least 25% of the chicken breast muscle can besubstituted for the protein isolate without any significant loss infunctional characteristics, with the exception of color.

Example 6 Alkaline Treatment of Animal Muscle Prevents Oxidation byInactivating Deoxyhemoglobin

To determined whether alkaline solubilization of animal muscle proteinled to advantages independent from enabling membrane lipid removal,washed cod muscle was prepared as described in Richards et al., J.Agric. Food Chem. 48:3141-3147, 2000. Trout hemolysate was then added tothe washed cod samples to achieve a hemoglobin concentration of 6μmol/kg. The samples were then stored at 5° C. for 15 hours afterestablishing a stable pH value for the sample. At the end of theincubation, thiobarbituric acid reactive substances (TBARS), a surrogatefor oxidation products, were quantified as described in Richards et al.,supra. The results are summarized in FIG. 1 and indicate thathemoglobin-dependent oxidation was reduced or eliminated at pH values ofabout 7 or above. At pH values below 7, significant oxidation wasobserved. In general, a TBAR value of greater than 20 μmol/kg indicatesstrong oxidation. As described in Richards et al., supra, this reductionin hemoglobin-dependent oxidation coincides with a decrease in theproportion of total hemoglobin that is in the form of deoxyhemoglobin.Thus, the results suggest that alkaline treatment of animal muscle,especially red animal muscle, prevents deoxyhemoglobin from reactingwith and oxidizing biological molecules in an animal muscle mixture,thereby explaining in part the benefits of the invention describedherein.

Example 7 Alkaline Treatment of Animal Muscle Improves Edible andGellable Protein Yield by at Least Two Mechanisms

To better understand the mechanism(s) for the high protein yields andgood quality gels described herein, herring muscle was prepared inhydrochloric acid (pH 2.6) as described in Kelleher et al., “Functionalchicken muscle isolates prepared using low ionic strength, andsolubilization/precipitation,” 53rd Ann. Reciprocal Meat Conf., Jun.18-21, 2000, Am. Meat. Sci. Assoc., Savoy, II, pp. 76-81. The sameprocedure was used to produce protein isolate in base (pH 10.7), exceptthat in this case, solubilization and incubation were done at analkaline pH using sodium hydroxide. The samples were incubated on icefor about 165 minutes and then loaded onto a 4-20% sodiumdodecylsulfate-polyacrylamide gel under standard reducing conditions.Electrophoresis of the gel and Coomassie Blue staining allowedvisualization of the myosin heavy chain protein band at about 205 kDa.Remarkably, the herring muscle incubated at a pH of 2.6 showedconsiderable breakdown of myosin heavy chain while no loss of myosinheavy chain was detected in the herring muscle incubated at a pH of10.7. It was hypothesized that alkaline conditions inhibited lysosomalproteases (e.g., cathepsins), which were responsible for myosinhydrolysis at a more acidic pH.

In a second experiment, frozen Alaskan pollock muscle protein wasprepared, solubilized under different pH conditions, and precipitated asdescribed in Example 1. The percentage by weight of protein recoveredafter precipitation was 22.7% at neutral pH, 66.1% at pH 11.0, and 58.5%at pH 3.0. It was noted that the protein recovered from the pH 11.0sample was able to form a gel. This result, in part, led to thefollowing hypothesis.

Gadoid fish, such as Alaskan pollock, Pacific hake, and blue whiting,are important food fish and are used to produce surimi. When gadoidspecies are frozen, an enzyme in the flesh, trimethylamine oxidedemethylase, breaks down triethylamine oxide in the flesh todimethylamine and formaldehyde. The formaldehyde produced in turndenatures muscle protein, thereby rendering them insoluble, even underalkaline conditions. It is believed that the alkaline treatmentdescribed can solubilize some of the modified proteins due to the highnegative charge on the proteins at this pH. It is also possible that thealkaline treatment can reverse at least in part the reaction of the fishproteins with formaldehyde, thereby rendering the fish protein soluble.

The results in this example suggest that the advantages of the presentinvention can operate through more than one mechanism.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the claims. Other aspects,advantages, and modifications are within the scope of the followingclaims.

1. A method for isolating edible protein from animal muscle, the methodcomprising obtaining animal muscle comprising animal muscle protein;preparing an animal muscle mixture comprising the animal muscle andwater, wherein the pH of the animal muscle mixture is sufficientlyalkaline to solubilize animal muscle protein; removing at least about50% by weight of total membrane lipids from the mixture; precipitatingthe animal muscle protein solubilized in the animal muscle mixture; andcollecting the precipitated protein, thereby isolating the protein fromthe animal muscle, wherein the temperature of the mixture is maintainedat 15° C. or less in each step of the method.
 2. The method of claim 1,wherein the collected precipitated protein is capable of forming anedible gel.
 3. The method of claim 1, further comprising forming anedible gel from the collected precipitated protein.
 4. The method ofclaim 1, wherein the animal muscle mixture is prepared by adding theanimal muscle to water to form a mixture and increasing the pH of themixture to a level sufficient to solubilize animal muscle protein in themixture.
 5. The method of claim 4, wherein the animal muscle comprisesabout 15% or less by weight of the mixture prior to increasing the pH ofthe mixture.
 6. The method of claim 4, wherein the animal musclecomprises about 10% or less by weight of the mixture prior to increasingthe pH of the mixture.
 7. The method of claim 4, wherein the pH of themixture is increased by adding polyphosphate to the mixture.
 8. Themethod of claim 4, wherein the mixture comprises a buffer prior toincreasing the pH of the mixture.
 9. The method of claim 8, wherein thebuffer is selected from the group consisting of glycine, arginine,asparagine, cysteine, dipeptide carnosine, taurine, pyrophosphate, andorthophosphate.
 10. The method of claim 1, wherein the animal musclemixture is prepared by adding an aqueous solution to the animal muscleto form a mixture, wherein the pH of the aqueous solution is at a pHsufficiently alkaline to solubilize animal muscle protein in themixture.
 11. The method of claim 1, wherein the pH of the animal musclemixture is about 10.0 or above.
 12. The method of claim 1, wherein thepH of the animal muscle mixture is about 10.5 or above.
 13. The methodof claim 1, wherein at least about 70% by weight of total membranelipids are removed from the mixture.
 14. The method of claim 1, whereinat least about 90% by weight of total membrane lipids are removed fromthe mixture.
 15. The method of claim 1, wherein membrane lipids areremoved by centrifugation of the mixture.
 16. The method of claim 15,wherein the mixture is centrifuged at about 5000×g or greater.
 17. Themethod of claim 15, wherein the mixture is centrifuged at about 7000×gor greater.
 18. The method of claim 15, wherein the mixture iscentrifuged at about 10,000×g or greater.
 19. The method of claim 1,wherein the membrane lipids are removed by filtration.
 20. The method ofclaim 1, wherein membrane lipids are removed by adding an aggregant tothe mixture.
 21. The method of claim 20, wherein the aggregant is apolymer.
 22. The method of claim 20, wherein the aggregant is a cationicpolymer.
 23. The method of claim 20, wherein the aggregant is an anionicpolymer.
 24. The method of claim 20, wherein the aggregant is selectedfrom the group consisting of carrageenan, algin, demethylated pectin,gum arabic, chitosan, polyethyleneimine, spermine, and spermidine. 25.The method of claim 20, wherein the aggregant is a salt.
 26. The methodof claim 20, wherein the salt is selected from the group consisting of acalcium salt, magnesium salt, sulfate, and phosphate.
 27. The method ofclaim 1, wherein the solubilized protein is precipitated by lowering thepH of the mixture.
 28. The method of claim 27, wherein the pH of themixture is lowered to about 5.5 or below.
 29. The method of claim 27,wherein the pH of the mixture is lowered to about 4.0 or below, thenraised to about 5.0 or above.
 30. The method of claim 27, furthercomprising adding a buffer to the mixture prior to precipitation of thesolubilized protein.
 31. The method of claim 30, wherein the buffer isselected from the group consisting of histidine, succinate, citrate,pyrophosphate, malonate, alanine, glutamic acid, citric acid, lacticacid, phosphoric acid, and pyruvic acid.
 32. The method of claim 1,wherein the precipitated protein is collected by centrifugation.
 33. Themethod of claim 1, wherein the solubilized protein is precipitated byadding an aggregant to the mixture after removal of membrane lipids. 34.The method of claim 33, wherein the aggregant is a polyamine.
 35. Themethod of claim 1, wherein the mixture comprises an iron chelator. 36.The method of claim 35, wherein the iron chelator is selected from thegroup consisting of ethylenediaminetetraacetic acid,diethylenetriaminepentaacidic acid, carnosine, anserine, uric acid,citric acid, phosphate, polyphosphate, ferritin, and transferrin. 37.The method of claim 1, further comprising washing the animal muscle withan aqueous solution prior to preparing the animal muscle mixture.
 38. Amethod of isolating edible protein from animal muscle, the methodcomprising obtaining animal muscle comprising animal muscle protein;preparing an animal muscle mixture comprising the animal muscle andwater, wherein the pH of the animal muscle mixture is increased to alevel sufficiently alkaline to solubilize animal muscle protein;precipitating the solubilized protein from the animal muscle proteinmixture; and collecting the precipitated protein, thereby isolating theedible protein from the animal muscle, wherein the temperature of themixture is maintained at 15° C. or less in each step of the method, andthe collected precipitated protein provides a yield of at least about70% by weight of the total animal muscle protein in the mixture prior toincreasing the pH.
 39. The method of claim 38, wherein the animal musclecomprises about 50% or less by weight of the mixture prior to increasingthe pH of the mixture.
 40. The method of claim 38, wherein the animalmuscle comprises about 30% or less by weight of the mixture prior toincreasing the pH of the mixture.
 41. The method of claim 38, furthercomprising removing insoluble matter from the mixture prior toprecipitation of the solubilized protein.
 42. The method of claim 41,wherein the insoluble matter is removed by centrifugation at about4000×g or below.
 43. The method of claim 38, wherein the animal musclemixture is prepared by adding the animal muscle to water to form amixture and increasing the pH of the mixture to a level sufficient tosolubilize animal muscle protein in the mixture.
 44. The method of claim43, wherein the animal muscle comprises membrane lipids, furthercomprising removing membrane lipids from the mixture after increasingthe pH of the mixture.
 45. The method of claim 44, wherein at leastabout 50% by weight of total membrane lipids are removed from themixture.
 46. The method of claim 38, wherein the animal muscle is fishmuscle.
 47. The method of claim 38, wherein the animal muscle is chickenmuscle.
 48. The method of claim 38, wherein the collected precipitatedprotein is capable of forming an edible gel.
 49. The method of claim 38,further comprising forming an edible gel from the collected precipitatedprotein.
 50. The method of claim 38, wherein the animal muscle mixtureis prepared by adding an aqueous solution to the animal muscle to form amixture, wherein the pH of the aqueous solution is at a pH sufficientlyalkaline to solubilize animal muscle protein in the mixture.
 51. Themethod of claim 38, wherein the animal muscle comprises membrane lipids,further comprising removing at least about 50% by weight of the totalmembrane lipids from the animal muscle mixture before precipitating thesolubilized protein.
 52. The method of claim 20, wherein the aggregantis a polyamine.